Methods for Tumor Diagnosis and Therapy

ABSTRACT

The present invention discloses a method for the enzyme-mediated, site-specific, in-vivo precipitation of a water soluble molecule in an animal. The enzyme is either unique to tumor cells, or is produced within a specific site (e.g., tumor) at concentrations that are higher than that in normal tissues. Alternatively, the enzyme is conjugated to a targeting moiety such as an antibody or a receptor-binding molecule.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/100,658, filed Dec. 9, 2013, now U.S. Pat. No. 9,183,425, which is acontinuation of U.S. patent application Ser. No. 13/459,698, filed Apr.30, 2012, now U.S. Pat. No. 8,603,437, which is a continuation of U.S.patent application Ser. No. 12/418,563, filed Apr. 3, 2009, now U.S.Pat. No. 8,168,159, which is a continuation of U.S. patent applicationSer. No. 09/839,779, filed Apr. 20, 2001, now U.S. Pat. No. 7,514,067,which claims the benefit of priority to U.S. Provisional PatentApplication No. 60/199,350, filed Apr. 25, 2000, the specification,claims, and drawings (if any) of all of which are hereby incorporated byreference into the specification of this application.

BACKGROUND OF THE INVENTION

Monoclonal antibodies (MAb), by virtue of their unique in-vitrospecificity and high affinity for their antigen, have generally beenconsidered particularly attractive as selective carriers of cancerradiodiagnostic/therapeutic agents. Several reasons underlie theseexpectations: (i) they show a high degree of specificity and affinityfor their intended target; (ii) they are generally nontoxic; and (iii)they can transport such agents. The application of MAb in animals andhumans for both tumor scintigraphic detection (labeled with ¹²³I, ¹³¹I,^(93m)Tc, and ¹¹¹In) and therapy (labeled with the beta emitters ¹³¹I,¹⁸⁶Re, ⁹⁰Y, ¹⁶⁵Dy, ⁶⁷Cu, and ¹⁰⁹Pd; the alpha emitters ²¹¹At, and ²¹²Bi,and ²¹³Bi; or conjugated to various toxins and cytotoxic drugs) is thefocus of work in many research laboratories.

In pursuing these studies, the basic assumption continues to be that MAbhave a role in the radioimmunodiagnosis and radioimmunotherapy ofcancer. However, while most published work on this subject hasdemonstrated their utility in the diagnosis and treatment of varioustumors in experimental animal models, the use of radiolabeled MAb totarget and treat solid tumors in cancer patients has been for the mostpart unsuccessful. There are at least five reasons for the results seenin humans:

-   1. Low Tumor Uptake. Thus far, most studies in humans have    demonstrated that the percentage injected dose per gram of tumor (%    ID/g) is extremely low. As a result, the absolute amount of the    therapeutic radionuclide within the tumor is much less than that    needed to deposit a radiation dose sufficiently high to sterilize    the tumor.-   2. High Activity in the Whole Body. A corollary to low tumor uptake    is the presence of ˜90%-99% of the injected radiolabeled MAb in the    rest of the body. This has led to the deposition of high doses in    normal tissues and unacceptable side effects, and a reduction in the    maximum tolerated dose (MTD).-   3. Slow Blood Clearance. In most human radioimmunotherapy trials,    whole MAb (MW ˜150,000 Da) have been used. The clearance of such    high-molecular-weight proteins from blood and nontargeted tissues is    rather slow. The resulting systemic exposure to the radioisotope    thus produces high doses to the bone marrow and a lowering of the    MTD.-   4. Limited Intratumoral Diffusion. The high molecular weight of MAb    also limits their ability to extravasate and diffuse through the    tumor mass. As a consequence, many areas within the tumor are spared    from receiving a lethal dose of radiation (i.e., the areas are    either outside the range of the emitted particle or receive a    sublethal dose).-   5. Heterogeneity of Tumor-Associated Antigen Expression. Many    studies have demonstrated that a substantial proportion of the cells    within a tumor mass show reduced/no expression of the targeted    antigen. This also will lead to nonuniform distribution of the    radionuclide within the tumor mass and the sparing of a large number    of cells within the tumor.

In an attempt to bypass some of the limitations of these uniquemolecules, various two-step and three-step approaches have beentheorized, in which a noninternalizing antitumor antibody is injectedprior to the administration of a low-molecular-weight therapeuticmolecule that has an affinity/reactivity with the preinjected antibodymolecule. These systems can be categorized into two major classes:MAb-directed enzyme prodrug therapy and MAb-directed radioligandtargeting, details of which are known in the art.

It is clear that under ideal conditions, a radiolabeled therapeuticagent must meet the following requirements: (i) be labeled with anenergetic particle emitter, (ii) be taken up rapidly and efficiently bythe tumor, (iii) be retained by the tumor (i.e. very long effectiveclearance half-life), (iv) have a short residence within normal tissues(i.e., short effective half-life in blood, bone marrow, and whole body),(v) achieve high tumor-to-normal tissue uptake ratios, (vi) attain anintratumoral distribution that is sufficiently uniform to match therange of the emitted particles (i.e. all tumor cells are within therange of the emitted particles), and (vii) achieve an intratumoralconcentration that is sufficiently high to deposit a tumoricidal dose inevery cell that is within the range of the emitted particle.

SUMMARY OF THE INVENTION

The present invention relates to a method for the enzyme-mediated,site-specific, in-vivo precipitation of a water soluble molecule in ananimal. The enzyme is either unique to tumor cells (i.e. only producedby tumor cells), or is produced within the specific site (e.g., tumor)at concentrations that are higher than that in normal tissues.Alternatively, the enzyme is conjugated to a targeting moiety such as anantibody. For example, an antibody-enzyme conjugate is injected intotumor bearing animals and following tumor targeting and clearance fromnormal tissues and organs, the water soluble substrate is injected.Owing to the negatively charged prosthetic group (e.g. phosphate)present within its molecules, the substrate is highly hydrophilic, isnot internalized by mammalian cells, and should clear from circulationat a rate that is compatible with its physical characteristics (e.g.molecular weight, charge). However, being a substrate for the enzyme(pre-targeted or otherwise), this water soluble molecule loses theprosthetic group and the resulting molecule precipitates out due to itshighly water-insoluble nature. The precipitated molecule is thus“indefinitely trapped” within the targeted tissue. In one of its aspects(Enzymatic Radiolabel Insolubilization Therapy, ERIT), the substrate isradiolabeled with a gamma or a positron emitting radionuclide and assuch, the location of the precipitate can be detected by externalimaging means (SPECT/PET). On the other hand, when the radionuclide isan alpha or a beta particle emitter, the trapped precipitatedradioactive molecule will maintain the radionuclide within the targetedtumor thereby enhancing its residence time and delivering a highradiation dose specifically to the tumor relative to the rest of thebody. In yet another aspect (Enzymatic Boron Insolubilization Therapy,EBIT), the substrate is conjugated to one/more boron-containing moleculeand upon precipitation within its intended target, the tumor issubjected to epithermal neutrons with the subsequent alpha particleemissions (Boron Capture Therapy).

In its simplest form, therefore, the present invention is based on theconversion of a chemical (e.g. quinazolinones, benzoxazoles,benzimidazoles, benzothiazoles, indoles, and derivatives thereof) from afreely water-soluble form to a highly water-insoluble form and hence invivo precipitation at the specific site where an enzyme (e.g.acetylglucosaminidases, acetylneuraminidases, aldolases,amidotranferases, arabinopayranosidases, carboxykinases, cellulases,deaminases, decarboxylases, dehydratases, dehydrogenase, DNAses,endonucleases, epimerases, esterases, exonucleases, fucosidases,galactosidases, glucokinases, glucosidases, glutaminases,glutathionases, guanidinobenzodases, glucoronidases, hexokinases,iduronidases, kinases, lactases, manosidases, nitrophenylphosphatases,peptidases, peroxidases, phosphatases, phosphotransferases, proteases,reductases, RNAses, sulfatases, telomerases, transaminases,transcarbamylases, transferases, xylosidases, uricases, urokinasess) orany other species capable of carrying out such a conversion in highconcentrations. Pretargeting of enzyme or its equivalent species may beachieved by making use of specific antibodies or any such specificreceptor-binding ligand to the desired sites in vivo. Note that theligand may also be a peptide or hormone, with the receptor specific tothe peptide or hormone.

Alternatively, the enzyme may be produced within the tumor site by thetumor cells themselves or following gene therapy or similar means. Thechemical to be injected in the second step contains any nuclide suitablefor imaging and/or therapy (e.g. Boron-10, Carbon-11, Nitrogen 13,Oxygen-15, Fluorine-18, Phosphorous-32, Phosphorous-33, Technetium-99m,Indium-111, Yttrium-90, Iodine-123, Iodine-124, Iodine-131,Astatine-211, Bismuth-212, etc.).

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate preferred embodiments of theinvention as well as other information pertinent to the disclosure, inwhich:

FIG. 1 is a graph of a time-course conversion of 1 (or Compound A) to 2(or Compound B) following incubation in alkaline phosphatase;

FIG. 2 is an illustration of the conversion of ¹²⁵I-1 (¹²⁵I-labeledCompound A) to ¹²⁵I-2 (¹²⁵I-labeled Compound B) following incubationwith ALP.

FIG. 3 is a graph of radioactivity following i.v. injection of ¹²⁵I-1into mice.

FIG. 4 is an illustration of the biodistribution of ¹²⁵I-1 in normalmice.

FIG. 5 is an illustration of the biodistribution of ¹²⁵I-2 in normalmice.

FIG. 6 is a graph of the accumulation of radioactivity within forelimbsof mice after subcutaneous (s.c.) injection of alkaline phosphatasefollowed by i.v. injection of ¹²⁵I-2.

FIG. 7 is a graph depicting the retention of radioactivity within aforelimb of mice injected s.c. with ¹²⁵I-1 or ¹²⁵I-2.

FIG. 8 is an illustration of the biodistribution (24 hours) ofs.c.-injected ¹²⁵I-2 in normal mice.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention describes a novel approach that serves to localizewater-insoluble, radioactive molecules within the extracellular(interstitial) space of a tumor. In one embodiment of this invention, anoninternalizing monoclonal antibody (MAb) to a “tumor-specific” antigenis chemically conjugated to the enzyme alkaline phosphatase (ALP); theMAb-ALP conjugate is administered intravenously (i.v.) to tumor-bearinganimals, and after MAb-ALP tumor localization and clearance fromcirculation (high tumor to normal tissue ratios), a water-soluble,radiolabeled prodrug (PD) that is a substrate for ALP is injectedintravenously. The conjugate or prodrug may also be injectedintra-arterially, subcutaneously, into the lymphatic circulation,intraperitoneally, intrathecally, intratumorally, intravesically, or isgiven orally.

The prodrug substrate is represented by the following formula:

R¹-D-(O-BLOCK)

-   wherein BLOCK is a blocking group that can be cleaved from the    remainder of the substrate by action of an enzyme, resulting in a    water-insoluble drug molecule represented by the following formula:

R¹-D-O—H

-   wherein D contains a minimum of 2 linked aromatic rings, and R¹ is a    radioactive atom, a molecule labeled with one or more radioactive    atom(s), a boron atom, or a molecule labeled with one or more boron    atoms.

The radiolabel is selected from the group consisting of a gamma emittingradionuclide suitable for gamma camera imaging, a positron emittingradionuclide suitable for positron emission tomography, and an alpha ora beta particle emitting radionuclide suitable for therapy. The alphaparticle emitting radionuclide may be, e.g., astatine-211, bismuth-212,or bismuth-213. The beta particle emitting radionuclide emits betaparticles whose energies are greater than about 1 keV. The beta particleemitting radionuclide may be, e.g., iodine-131, copper-67, samarium-153,gold-198, palladium-109, rhenium-186, rhenium-188, dysprosium-165,strontium-89, phosphorous-32, phosphorous-33, or yttrium-90. Note alsothat the boron atom is suitable for neutron activation.

The BLOCK is selected from the group consisting of:

-   a monovalent blocking group derivable by removal of one hydroxyl    from a phosphoric acid group, a sulfuric acid group, or a    biologically compatible salt thereof;-   a monovalent blocking group derivable by removal of a hydroxyl from    an alcohol or an aliphatic carboxyl, an aromatic carboxyl, an amino    acid carboxyl, or a peptide carboxyl; and-   a monovalent moiety derived by the removal of the anomeric hydroxyl    group from a mono- or polysaccharide.

As the PD molecules percolate through the tumor mass, they will behydrolyzed by the ALP molecules present within the tumor (MAb-ALP). Thehydrolysis of PD (Compound 1, or Compound A below) leads to theformation of a water-insoluble, radiolabeled precipitate (D). It isanticipated that D (Compound 2, or Compound B below), as a consequenceof its physical properties, will be trapped within the extracellularspace of the tumor mass. Thus, when labeled with iodine-131 (¹³¹I), aradionuclide that decays by the emission of both a beta particle(E_(max)=610 keV; mean range=467 μm; maximum range=2.4 mm) and photonssuitable for external imaging, the entrapped ¹³¹I-labeled D moleculeswill serve as a means for both assessing tumor-associated radioactivity(planar/SPECT) and delivering a protracted and effective therapeuticdose to the tumor.

Radiolabeled 2-(2′-hydroxyphenyl)-4-(3H)-quinazolinone dyes are employedin the method of the present invention: These classes of compoundscontain a hydroxyl group that forms an intramolecular six-memberedstable hydrogen bond with the ring nitrogen and hence they are highlywater-insoluble in nature. However, addition of a prosthetic group (e.g.phosphate, sulfate, sugars such as galactose or peptide) on the hydroxygroup renders the molecule freely water-soluble. Furthermore, thepresence of such prosthetic groups makes cell-membranes impermeable tothese molecules; they are anticipated to have relatively shortbiological half-lives in the blood. However, when acted on by theenzyme, the prosthetic group is lost, resulting in the restoration ofintramolecular hydrogen bonding, and the molecule becomeswater-insoluble and precipitates.

The procedure for the synthesis of the unsubstituted quinazolinone dye(1, below), is as follows:

1.3 g anthranilamide (3) and 1.2 g salicylaldehyde (4) were refluxed inmethanol. Within 30 minutes, a thick orange precipitate of theSchiff-base (5) was formed. The reaction mixture was cooled in arefrigerator and the product filtered and washed with cold methanol. Theprecipitate (about 1.5 g) was then suspended in 20 ml ethanol containingp-toluene sulfonic acid and refluxed for 1 hour. The progress ofreaction was followed by TLC. The off-white precipitate ofdihydroquinazolinone (6) was filtered off and washed thoroughly withcold ethanol. It was then suspended in 12 ml methanol containing 0.6 gdichloro-dicyanobenzoquinone and heated under reflux for 1 hour. Thequinazolinone dye product (2) was isolated after cooling the reactionmixture and subsequent filtration. The pale yellow chemical wassuspended in diethyl ether and stirred, filtered and washed with ether.To 100 mg of the quinazolinone dye (2) in 1 ml of dry pyridine undernitrogen in an ice bath was added 65 mg (40 μl) of POCl₃ through asyringe. After stirring at this temperature for 30 minutes, it wasneutralized by the addition of 116 μl of 30% ammonia. The crude product(1) was evaporated to dryness in rotary evaporator and was partitionedbetween ethyl acetate and water, the organic layer re-extracted withwater, and the combined aqueous extract was back extracted with ethylacetate. Finally, the product was loaded on to a DEAE Sephacel column(10 ml) pre-equilibrated in bicarbonate form. The column was washed with20 ml water followed by a stepwise gradient of triethyl ammoniumbicarbonate buffer (pH 7.0, 0.1 M to 0.5 M, 25 ml each). Appropriatefluorescent fractions were pooled and lyophilized to dryness (yield55%). All chemicals were characterized by NMR and elemental analysis.

In order to make use of the above chemistry for the synthesis ofradiolabeled quinazolinones, halogen substituted anthranilamides thatare easily converted to the tin prescursors needed for the exchangelabeling with radiohalogens were used. Thus, for the synthesis of5-haloanthranilamides (9) from 5-haloanthranillic acids (7), isatoicanhydrides were used, as shown below.

Such anhydrides are known to react with amines to furnishanthranilamides under certain controlled conditions. Since phosgene gasis not available, reaction conditions were employed using triphosgenewhich is a solid. 5-Halo-anthranilic acid (bromo- or iodo-) was stirredwith equimolar amounts of triphosgene in dry THF at ambient temperaturefor 1-2 hours. The solution was filtered and diluted with hexane untilit became turbid and then stored at −20° C. overnight. The precipitatedanhydride (8) was filtered and washed copiously with hexane THF mixtureand dried (yield ˜60%). The anhydride (dissolved in THF) was stirred in1 M aqueous solution of ammonia (containing 1:1 THF) at ambienttemperature for 25 minutes. Finally, the organic layer was evaporatedunder nitrogen and the product (9) was filtered and washed copiouslywith water followed by acetonitrile and dried in vacuo (yield ˜75%).

2-Amino-5-iodobenzoic acid (10) and triphosgene were then dissolved indry THF and the reaction mixture stirred at room temperature for 1 hour.An off-white precipitate formed and TLC showed that compound 10 isconsumed, as shown below. The precipitate was filtered, washed with coldmethanol, and crystallized in acetonitrile. ¹H NMR indicated that thespectrum was an iodoisotoic anhydride (11).

A solution of iodoisotoic anhydride (11) was then suspended in THF andcooled in an ice-bath. Aqueous ammonium hydroxide was added dropwise,the reaction mixture was stirred for 15 minutes at 0° C. and 30 minutesat RT, and the solvent was evaporated. The white solid obtained wascharacterized by ¹H NMR and identified as an iodoanthranilamide (12).

Next, Iodoanthranilamide (12) and salicylaldehyde were suspended inmethanol and refluxed in the presence of catalytic amounts of p-toluenesulfonic acid (TsOH) for 30 minutes. To the pale-yellow precipitate (13)formed, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone was added and thesuspension was refluxed for 1 hour. The solid product was filtered,washed with cold methanol, characterized by ¹H NMR, and identified as2-(2′-Hydroxyphenyl)-6-iodo-4-(3H)-quinazolinone.

Synthesis of Ammonium2-(2′-Phosphoryloxyphenyl)-6-iodo-4-(3H)-quinazolinone (15)

In one method, 2-(2′-Hydroxy)-6-iodo-4-(3H)-quinazolinone (14) was addedto dried pyridine at 0° C., followed by phosphorus oxychloride. Silicagel TLC indicated that the reaction was completed within 2 min. Thereaction solution was neutralized to pH 7.0 by the addition of ammoniumhydroxide. The solvent was evaporated and the solid product wassuspended in water, filtered, and purified by chromatography. Followingelution (stepwise gradient: water followed by acetonitrile-water, 2:1),a yellow solution containing UV-visible product was collected, thesolvent was evaporated, and the product was characterized by ¹H and ³¹PNMR and identified as compound 15.

In an alternative method, ammonium2-(2′-phosphoryloxyphenyl)-6-tributylstannyl-4-(3H)-quinazolinone (17)was dissolved in methanol, and sodium iodide was added followed byhydrogen peroxide. A yellow precipitate formed immediately. The reactionvial was vortex-mixed and incubated for 30 minutes at 37° C.Reversed-phase silica gel TLC showed approximately 50% conversion(solvent: acetonitrile-water, 1:1). The solvent was evaporated and theproduct purified by chromatography. TLC (solvent: chloroform-methanol,1:1) showed the same R_(f) value (0.6), and proton and ³¹P NMR gave thesame spectra as were obtained with the known compound 15 synthesized bythe route shown above.

Next, to a dioxane solution containing 14, hexa-n-butylditin andtetrakis (triphenylphosphine) palladium were added, as shown above. Thereaction mixture was refluxed for 1.5 hours and progress of the reactionwas followed by silica gel TLC (solvent: methylene chloride-ethylacetate, 9:1) to test for the formation of a more nonpolar product. Thesolvent was evaporated, and the crude yellow solid was purified on asilica gel column (stepwise gradient: starting with hexane followed byhexane-dichloromethane, 1:1). Following solvent evaporation, a yellowfluorescent solid 2-(2′-Hydroxy)-6-tributylstannyl-4-(3H)-quinazolinone16 was obtained as identified by ¹H NMR.

Next, to a stirred solution of 16 in dry pyridine cooled to 0° C.,phosphorus oxychloride was added dropwise. The reaction mixture wasstirred for 10 min at 0° C. and then quenched by the addition ofammonium hydroxide (Scheme 5). The solvent was evaporated, the crudeproduct redissolved in methanol-acetate (1:1) and purified on a C₁₈column (stepwise gradient: water followed by acetonitrile-water goingfrom 30% to 50% acetonitrile). The solvent was evaporated and thenonfluorescent solid Ammonium 2-(2′-Phosphoryloxyphenyl)6-tributylstannyl-4-(3H)-quinazolinone 17 was obtained as identified by³¹P NMR.

Next, three Iodo-beads were placed in a reaction vial, followed by 20 μlof 1 μg/μl solution of 17, 30 μl 0.1 M borate buffer (pH=8.3), andNa¹²⁵I (800 μCi/8 μl of 0.1 M sodium hydroxide). After 20 minutes atroom temperature, the crude reaction mixture was loaded on a Sep-PakPlus C₁₈ cartridge and eluted with 1 ml water and then 2 ml 10%acetonitrile in water. The product 18 was eluted with 20% acetonitrilein water (yield: ˜370 μCi; radiochemical yield: 46%). The radiolabeledproduct, co-spotted with nonradioactive compound 15 on reversed-phaseTLC, showed a single spot on autoradiograph (solvent:acetonitrile-water, 1.5:2). Radiolabeled (¹⁵¹I) Ammonium2-(2′-Phosphoryloxyphenyl)-6-iodo-4-(3H)-quinazolinone 18 co-injectedwith 15 into the HPLC showed a single radioactive peak (R_(f)=14 min)which matched the R_(f) value of 15.

X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactose) is routinely used forthe identification of lac⁺ bacterial colonies. The underlying principleis that the colorless X-gal which is freely water-soluble is convertedto dark blue colored precipitate upon reaction with b-galactosidaseenzyme.

In accordance with the present invention, a bromine atom within X-gal isreplaced with a diagnostic/therapeutic nuclide (e.g. Iodine-131,boron-10). For example, an antibody-β-galactosidase immunoconjugate willbe administered in the first step. Following its clearance, the watersoluble nuclide-labeled X-gal (10) will be injected where at the sitesof enzyme action, the X-gal substrate will lose the sugar moiety and theresulting aglycon (11) will precipitate within the targeted tissue. Inanother embodiment, prosthetic groups other than galactosyl (e.g.phosphate, sulfate, carbonate) as well as nuclides other than iodine-131and boron-10 may be used.

When the prodrug ((1) or Compound A), a non-fluorescent, stable,water-soluble compound, is incubated (37° C.) with alkaline phosphatase(ALP), a bright yellow-green fluorescent, clearly visible precipitate isformed whose R_(f) on thin layer chromatography (TLC) corresponds todrug ((2) or Compound B). In order to assess the kinetics of thisenzyme-based hydrolysis (i.e. conversion of Compound A to Compound B) at37° C., Compound A (60 μM) was mixed with 10 units ALP in 0.1 M Tris (pH7.2) and the reaction kinetics followed over time using a Perkin-ElmerLS50B Luminescence Spectrometer with excitation at 340 nm and emissionat 500 nm. There is a rapid increase in fluorescence intensity underthese experimental conditions, as shown in FIG. 1, demonstrating thehydrolysis of Compound A and the formation of Compound B. Nofluorescence was observed when the enzyme was heat-inactivated prior toits incubation with Compound A.

In order to further characterize the ¹²⁵I-labeled prodrug, ¹²⁵I-labeledCompound A (˜10 μCi/100 μl 0.1 M Tris buffer, pH 7.2) was incubated with5 units ALP or heat-inactivated (70° C., 2 hours) ALP; the samples werespotted on reversed-phase TLC plates that were then run inacetonitrile-water (1.5:2). Autoradiography demonstrates the completeconversion of ¹²⁵I-labeled Compound A to ¹²⁵I-labeled Compound B only inthe presence of the active enzyme, as shown in FIG. 2.

In order to determine blood clearance of AP¹²⁵IQ, mice (n=5/group) wereinjected i.v. with the radioiodinated prodrug and bled over a 1 hourperiod, the radioactive content per gram of blood was measured, and thepercentage injected dose per gram (% ID/g) calculated. The results asshown in FIG. 3 demonstrate a rapid biphasic blood clearance ofradioactivity and a T_(1/2β) of 51.1±6.8 min.

In order to assess the chemical nature of the radioactivity in blood(i.e. determine stability of ¹²⁵I-labeled Compound A in blood), ethanolwas added to the blood samples (collected during the first 40 min), thetubes were centrifuged, and the supernatant was spotted on TLC. Theplates were run in acetonitrile-water (1.5:2) and autoradiographed. Theresults show (i) the presence of a single spot whose R_(f) is the sameas that observed with ¹²⁷I-labeled Compound A, and (ii) no evidence offree iodine. These data demonstrate the stability of I-labeled CompoundA in serum.

The biodistribution of ¹²⁵I-labeled Compound A in normal tissues wasalso considered. Mice (n=30) were injected i.v. with theradiopharmaceutical (˜5 μCi/100 μl), the animals were killed at 1 hour(n=15) and 24 hours (n=15), and the radioactivity associated with blood,tissues, and organs was determined. As shown in FIG. 4, (i) theradioactivity in all organs and tissues declined over time; (ii) theradioactivity in the kidneys and urine was high, suggesting that thecompound and/or its metabolic breakdown/hydrolysis products were rapidlyexcreted (since the weight of the thyroid is ˜5 mg, the activity withinthe thyroid indicates minimal dehalogenation of the compound); and (iii)<20% of the injected dose remained in the body by 24 hours. Theseresults, therefore, demonstrate that ¹²⁵I-labeled Compound A and/or itsmetabolic breakdown/hydrolysis product(s) have a low affinity to normaltissues and that the presence of endogenous ALP leads to minimalhydrolysis of the compound.

The biodistribution of ¹²⁵I-labeled Compound B was also examined invarious tissues in normal mice. In these experiments, ¹²⁵I-labeledCompound A was synthesized, purified, and incubated at 37° C. in thepresence of ALP overnight. TLC demonstrated the complete conversion of¹²⁵I-labeled Compound A to ¹²⁵I-labeled Compound B. Mice (n=10) wereinjected i.v. with ¹²⁵I-labeled Compound B (˜5 μCi/100 μl) and killed(n=5) at 1 hour and 24 hours. The 1 hour data, as shown in FIG. 5,demonstrate the presence of this water-insoluble molecule in all thetissues examined (<15% ID/g). However, by 24 hours (FIG. 5), all tissuesand organs (with the exception of minimal activity in the thyroid) werevirtually void of radioactivity (<4% of the injected dose remained inthe body by 24 hours). These results show that ¹²⁵I-labeled Compound Bhas no avidity for any tissue in the mouse and that thetissue-associated activity seen at 1 hour reflects that within theblood. Since these data seem to argue for the inability of ¹²⁵I-labeledCompound B to traverse blood vessel walls and enter into tissues, thiswater-insoluble molecule if formed within a tissue (e.g. tumor mass) islikely to be retained, i.e. it will not leach back into circulation.

In order to demonstrate within an animal the conversion of thewater-soluble ¹²⁵I-labeled Compound A to the water-insoluble¹²⁵I-labeled Compound B, ALP was dissolved in saline (50, 100, 150, 200,250, 300, 400 units/10 μl) and using a 10-μl syringe, 10 μl enzymepreparation was injected s.c. in the forelimb of Swiss Alpine mice.Five-minutes later, 20 μCi ¹²⁵I-labeled Compound A was injected i.v.(tail vein). The animals were killed 1 hour later and the radioactivityin the forelimbs was measured. The results, as shown in FIG. 6,demonstrate that the radioactive content within the forelimbs of animalspre-injected with ALP increased with enzyme dose and plateaued at thehighest concentrations. The fact that these increases were due to theenzymatic action of ALP was ascertained in studies that showed noincrease in uptake in the forelimbs of mice pre-injected withheat-inactivated ALP (FIG. 6). These results illustrate the specificdose-dependent accumulation of ¹²⁵I-labeled Compound A (more accurately,¹²⁵I-labeled Compound B) within alkaline-phosphatase-containing sites inan animal.

In order to demonstrate that once formed, the water-insoluble Compound Bis retained “indefinitely” within the tissue where it is formed,¹²⁵I-labeled Compound B was dissolved in 100 μl DMSO (under theseconditions, ¹²⁵I-labeled Compound B is completely soluble in DMSO;however, when 100 μl water are added, a visible precipitate formsimmediately that contains ¹²⁵I-labeled Compound B radioactivity). Fiveμl of this solution was injected s.c. into the right forelimb of mice(n=15), followed by 5 μl saline. For comparison, ¹²⁵I-labeled Compound A(5 μCi/5 μl saline) was injected s.c. into the left forelimb of the samemice and followed with 5 μl DMSO. The animals were killed after 1 hour,24 hours, and 48 hours, the radioactivity associated with the forelimbswas measured, and the percentage of radioactivity remaining wascalculated (at the 24 hour time point, the biodistribution ofradioactivity in various tissues and organs was also determined). Thedata (FIG. 7) demonstrate that while greater than about 98% of theprodrug ¹²⁵I-labeled Compound A had seeped out of the s.c. pocket by 24hours, 71±5% of the injected precipitable ¹²⁵I-labeled Compound Bactivity remained at the injection site at 24 hours. The biodistributiondata (FIG. 8) show that the radioactivity that escaped during the first24 hours following the s.c. injection of ¹²⁵I-labeled Compound B doesnot localize in any normal tissues within the animal (activity withinthe thyroid indicates uptake of free iodine). Finally, the results showno change in the radioactivity in the forelimbs of the animals at 24hours and 48 hours (FIG. 7), thereby indicating that the precipitated¹²⁵I-labeled Compound B is permanently and indefinitely trapped withintissues.

While this invention has been described in terms of exemplaryembodiments, it is not limited thereto. Rather, the appended claimsshould be construed broadly to include other variants and embodiments ofthe invention which may be made by those skilled in the art withoutdeparting from the scope and range of equivalents of the invention.

1.-20. (canceled)
 21. A method for the enzyme-mediated, site-specific,in-vivo localization of a water-insoluble molecule at a tumor site,comprising: administering to an animal a water-soluble prodrug having astructure:

or a biologically compatible salt form thereof, wherein R₁ is aradioactive atom or a radiolabeled moiety comprising one or moreradioactive atoms, R₂ is hydrogen, and R₃ is a prosthetic group that iscleavable by an enzyme that is present at the tumor site, and by virtueof said administering, the prodrug is hydrolyzed by the enzyme to form awater-insoluble drug at the tumor site.
 22. The method of claim 21,wherein the water-soluble prodrug has the structure:


23. The method of claim 22, wherein R₁ is selected from the groupconsisting of a gamma emitting radionuclide, a positron emittingradionuclide, and an alpha or a beta particle emitting radionuclide. 24.The method of claim 22, wherein R₁ is selected from the group consistingof astatine-211, bismuth-212, bismuth-213, bromine-77, iodine-123,iodine-124, iodine 125, iodine-131, copper-67, samarium-153, gold-198,palladium-109, rhenium-186, rhenium-188, dysprosium-165, strontium-89,phosphorous-32, phosphorous-33, and yttrium-90.
 25. The method of claim24, wherein R₁ is radioactive iodine.
 26. The method of claim 22,wherein R₃ is selected from phosphate, sulfate, carbonate, andgalactosyl.
 27. The method of claim 26, wherein R₃ is phosphate.
 28. Themethod of claim 25, wherein R₃ is phosphate.
 29. The method of claim 22,wherein said administering is by injection.
 30. The method of claim 28,wherein said injection is carried out intravenously, intra-arterially,subcutaneously, into the lymphatic circulation, intraperitoneally,intrathecally, intratumorally, or intravesically.
 31. The method ofclaim 22, wherein said animal is a human.
 32. A compound having astructure:

or a biologically compatible salt form thereof, wherein R₁ is aradioactive atom or a radiolabeled moiety comprising one or moreradioactive atoms, R₂ is hydrogen, and R₃ is a prosthetic group that ishydrolytically cleavable by an enzyme.
 33. The compound of claim 32,having the structure:


34. The compound of claim 33, wherein R₁ is selected from the groupconsisting of a gamma emitting radionuclide, a positron emittingradionuclide, and an alpha or a beta particle emitting radionuclide. 35.The compound of claim 34, wherein R₁ is selected from the groupconsisting of astatine-211, bismuth-212, bismuth-213, bromine-77,iodine-123, iodine-124, iodine 125, iodine-131, copper-67, samarium-153,gold-198, palladium-109, rhenium-186, rhenium-188, dysprosium-165,strontium-89, phosphorous-32, phosphorous-33, and yttrium-90.
 36. Thecompound of claim 35, wherein R₁ is radioactive iodine.
 37. The compoundof claim 33, wherein R₃ is selected from phosphate, sulfate, carbonate,and galactosyl.
 38. The compound of claim 37, wherein R₃ is phosphate.39. The compound of claim 36, wherein R₃ is phosphate.
 40. The compoundof claim 32 comprised in a formulation suitable for injection.
 41. Thecompound of claim 33 comprised in a formulation suitable for injection.